Abstract
Uterine leiomyomas, benign uterine smooth muscle tumors that affect 30% of reproductive-aged women, are a significant health concern. The initiation event for these tumors is unclear, but 17β-estradiol (E2) is an established promoter of leiomyoma growth. E2 not only alters transcription of E2-regulated genes but also can rapidly activate signaling pathways. The aim of our study is to investigate the role of rapid E2-activated cytoplasmic signaling events in the promotion of leiomyomas. Western blot analysis revealed that E2 rapidly increases levels of phosphorylated protein kinase Cα (PKCα) in both immortalized uterine smooth muscle (UtSM) and leiomyoma (UtLM) cell lines, but increases levels of phosphorylated ERK1/2 only in UtLM cells. Our studies demonstrate a paradoxical effect of molecular and pharmacological inhibition of PKCα on ERK1/2 activation and cellular proliferation in UtLM and UtSM cells. PKCα inhibition decreases levels of phosphorylated ERK1/2 and proliferation in UtLM cells but raises these levels in UtSM cells. cAMP-PKA signaling is rapidly activated only in UtSM cells with E2 and inhibits ERK1/2 activation and proliferation. We therefore propose a model whereby E2’s rapid activation of PKCα and cAMP-PKA signaling plays a central role in the maintenance of a low proliferative index in normal uterine smooth muscle via its inhibition of the MAPK cascade and these pathways are altered in leiomyomas to promote MAPK activation and proliferation. These studies demonstrate that rapid E2-signaling pathways contribute to the promotion of leiomyomas.
Estradiol-17β initiates rapid signaling events that promote the growth of uterine leiomyoma cells but inhibits the growth of normal uterine smooth muscle cells.
Uterine leiomyomas, or fibroids, are benign uterine smooth muscle tumors and are a significant women’s health issue due to their high incidence and morbidity. Whereas one third of all premenopausal women are symptomatic for uterine leiomyomas, it is suspected that up to 80% of women develop these tumors (1,2). Uterine leiomyomas can lead to abnormal bleeding, pregnancy complications, and infertility and are a leading cause of hysterectomies (3).
Although predisposing factors for the development of uterine leiomyomas exist and include ethnicity (2,4), early use of oral contraceptives (5,6), prenatal exposure to diethylstilbestrol (7,8,9,10), and obesity (11), the initiation event for these tumors is unclear and may be due to genetic or epigenetic alterations (12,13,14,15,16,17,18,19). It is clear, however, that enhanced proliferation is a primary factor in uterine leiomyoma tumor growth (20,21). Moreover, it is generally accepted that estrogens are an important driving force behind this increased proliferation (22,23,24). Evidence for the dependence of leiomyomas on the steroid hormone 17β-estradiol (E2) include: 1) the onset of leiomyomas only after puberty; 2) the regression of leiomyoma tumors with a reduction in circulating E2 levels as a result of menopause or treatment with GnRH agonists (25); 3) the decrease in E2-induced proliferation of leiomyomas with estrogen receptor (ER) antagonists, including selective ER modulators (23,26,27); and 4) the inhibition of leiomyoma growth in mice with inactive or down-regulated ERs (28,29). The role of progesterone in leiomyoma growth is less clear. Progesterone has been shown to up-regulate Bcl-2, an antiapoptotic factor, in leiomyoma cells (30) but can also lower factor signaling (31) to reduce E2-stimulated leiomyoma growth. A reduced proliferative signaling with progesterone may help account for the protective effect that pregnancy can have on leiomyoma expansion (32).
Uterine leiomyomas arise from the smooth muscle layer of the uterus, which is normally quiescent. In early gestation, the smooth muscle cells of the uterus demonstrate a dramatic increase in proliferation in response to the hormonal cues such as increasing levels of E2 and human chorionic gonadotropin (33). A question of interest that has not yet been investigated is what prevents normal uterine smooth muscle cells from proliferating in the face of similar hormonal cues (i.e. estrogens) in the nonpregnant state? Also, can these processes provide clues as to what is altered in leiomyomas?
Estrogens may exert effects on normal uterine smooth muscle and leiomyoma cells through several mechanisms. In addition to altering transcription of E2-regulated genes, E2 can rapidly initiate cytoplasmic signaling pathways in a variety of cell types. As reviewed by many authors, various signaling molecules, including protein kinases and G protein-coupled receptors, can be activated by E2 within minutes, depending on the cellular context (34,35,36,37,38,39,40). The rapid activation of these signaling cascades ultimately converge at the nucleus to affect gene transcription (41,42,43,44).
Genetic studies of uterine leiomyomas have not discovered key altered genes and expression of E2-regulated genes such as c-myc, cyclin D1, and pS2 is not as high as expected for a disease that is driven by E2 (45,46,47,48,49,50,51,52). As demonstrated in breast cancer cells, high levels of rapid E2 signaling may exist, even without a robust genomic estrogenic response in classic E2-responsive genes (53). Several studies suggest that growth factor signaling is important to leiomyoma growth (50,54,55,56,57). Rapid E2 and growth factor signaling can converge because both E2 and growth factors can trigger multiple pathways, including MAPK, to promote cell cycle progression in several cell types (58,59). Moreover, rapid E2 signaling and growth factor signaling cross talk has been reported in E2-dependent breast cancer and can act synergistically to stimulate cell proliferation (60). To date, there is no detailed knowledge of how rapid E2 signaling may be contributing to leiomyoma growth.
In this study, we investigated the contribution of rapid E2 signaling events to both the proliferation of leiomyoma cells and the maintenance of low levels of proliferation in normal uterine smooth muscle cells. Our results demonstrate that E2 activates multiple signaling pathways in these cell types that converge on MAPK to modulate the cells proliferative response to E2. We report that cAMP-protein kinase A (PKA) signaling is initiated by E2 and has an inhibitory effect on MAPK signaling and proliferation in the normal uterine smooth muscle but that this pathway is absent in the leiomyoma cells. Moreover, E2’s elevation of phospho-protein kinase C (PKC)-α levels and PKCα’s paradoxical effects on ERK1/2 activation play a pivotal role in the pathological growth of uterine leiomyoma cells and in the repression of proliferation in normal uterine smooth muscle cells.
Materials and Methods
Cell culture
Uterine leiomyoma (UtLM-ht) and normal uterine smooth muscle (UtSM-ht) cell lines, immortalized via retroviral transfection (PLX1N vector; CLONTECH, Palo Alto, CA) of human telomerase, were graciously donated by Darlene Dixon (National Institute of Environmental Health Sciences; Research Triangle Park, NC). Cells were maintained in DMEM containing 10% fetal bovine serum (FBS; Invitrogen Life Technologies; Carlsbad, CA) supplemented with 1% essential and nonessential amino acids, 1% sodium pyruvate, and 0.14% geneticin (30 mg/ml).at 37 C in a humidified atmosphere of 5% CO2.
In vivo xenograft studies
Twenty ovariectomized female nude mice (ν/ν) mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA). Ten mice were sc injected with 10 × 106 UtLM-ht cells and ten were sc injected with 5 × 106 UtSM-ht cells in each dorsal flank. Five of the UtLM-ht and UtSM-ht injected mice were given E2 pellets (0.72 mg per 60 d release; Innovative Research of America, Sarasota, FL), and the other five were given blank pellets. Tumors were measured biweekly and animals were killed at d 25. Tumor volume and weight was then measured.
All procedures involving these animals were conducted in compliance with state and federal laws, standards of the United States Department of Health and Human Services and guidelines established by Tulane University Animal Care and Use Committee. The facilities and laboratory animals program of Tulane University are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care.
RT-PCR
Total RNA was extracted from treated cells using an RNAeasy mini kit (QIAGEN, Valencia, CA) according to the manufacturer’s instructions. Total RNA (2 μg) was reversed transcribed using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA). Aliquot of 10× diluted cDNA, iQ SYBR Green (Bio-Rad), and specific primers were amplified using a Bio-Rad iCycler iQ real-time PCR detection system. Primers were designed using Beacon Designer 2 software (Palo Alto, CA) and ordered through Invitrogen primer division. Primers were: progesterone receptor (TACCCGCCCTATCTCAACTACC) and β-actin (TGAGCGCGGCTACAGCTT). PCR parameters were denature at 95 C for 3 min followed by 40 cycles of 95 C for 20 sec and annealing/extension at 60 C for 1 min. Melt curves were generated to confirm each primer and data were analyzed by δ-quantitative comparative threshold value method as determined by the iCycler software system.
Western blot analysis
Briefly, cells were washed and phenol-free DMEM containing 5% charcoal-stripped FBS was added to the cells 72 h before treatment. Cells were then pretreated with 0.1% ethanol, 1 μm ICI 182, 780 (Tocris, Ellisville, MO), 5 μm H-89, 10–100 nm Ro 31,8220 (Sigma, St. Louis, MO), or 100 pm-15 nm bisindolymaleimide × hydrochloride (Bis10) (Sigma) and then treated with 1 nm E2 (Sigma; St. Louis, MO) for 5 min. Dosage ranges for the inhibitors were calculated based on specificity to PKCα and PKA and toxicity. Cells were immediately lysed and protein was resolved by SDS-PAGE on a 4–12% gradient gel. Protein was transferred to nitrocellulose membranes and the membranes were incubated with specific antibodies (Cell Signaling Technology, Boston, MA) at 4 C. Membranes were then incubated first with antirabbit IgG conjugated to horseradish peroxidase and then with a chemiluminescent substrate (Pierce, Rockford, IL) to detect antibody-antigen complexes. Densitometry of the bands was used for quantification, and results of at least three experiments were combined for statistical analysis.
Transfection of constitutive-active and dominant-negative PKCα
Plasmid DNA, graciously donated by Barbara Beckman (Tulane University, New Orleans, LA), was introduced to cells maintained for 72 h in phenol-free DMEM containing 5% charcoal-stripped FBS by nucleofection (AmaxaBiosystems, Gaithersburg, MD; primary smooth muscle kit). Transfected cells were allowed to adhere overnight in six-well plates and then treated and harvested as described above for Western blot analysis. Experiments were repeated three times.
Proliferation assays
Cells were changed to phenol-free DMEM containing 5% charcoal-stripped FBS 72 h before plating. Then 2 × 103 cells/well were seeded onto 48-well plates, and 18 h after plating, medium was changed to phenol-free DMEM containing 5% charcoal-stripped FBS and one of the following treatments: 0.1% ethanol, 1–10 nm E2 (Sigma), 1 μm ICI 182, 780 (Tocris), 50 nm Ro 31, 8220 (Sigma), 50 μm 3-isobutyl-1-methylxanthine (IBMX; Sigma), 100 μm 8-bromoadenosine-3′,5′cyclic monophosphate (8brcAMP; Sigma), or 100 pm Bis 10 (Sigma). Cells were maintained in treatment medium until proliferation was measured using the CellTiter 96 AQueous nonradioactive cell proliferation assay kit [Promega, Madison, WI; 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay]. Absorbance of the formazan product was measured on a FL600 microplate reader (Bio-Tek, Winsookie, VT). Experiments were repeated at least three times.
cAMP assays
cAMP levels with vehicle, E2 (Sigma), or 100 nm Ro 31,8220 (Sigma) were assessed with the use of a cAMP competitive EIA kit (Zymed Laboratories, Invitrogen, Carlsbad, CA). Briefly, cell media were changed to 5% charcoal-stripped FBS 72 h before treatment. Cells were then treated for the specific time increments. Cells were washed with PBS and flooded with 5% trichloroacetic acid, followed by a water wash. Supernatants were then incubated with enzyme-labeled cAMP and antibody. The reaction was then stopped and absorbance read using a Bio-Tek FL 600 microplate reader at 410 nm. cAMP concentration was calculated using a standard curve. Assay was completed in triplicate.
Statistical analysis
Prism 3.0 software (GraphPad, San Diego, CA) was used to conduct a one-way ANOVA with Tukey posttest for the data sets. Confidence interval was set at 95%.
Results
Genomic signaling and proliferative response in leiomyoma cells with E2 treatment
RT-PCR of progesterone receptor expression, an E2-responsive gene, demonstrates that genomic E2 signaling is intact in both UtSM-ht and UtLM-ht cells but is higher in the UtLM-ht cells (Fig. 1A). This is in agreement with studies that demonstrate that leiomyomas have a greater transcriptional response to E2 compared with the adjacent myometrium (61).
A hallmark of leiomyoma cells is their increased sensitivity to the mitogenic effects of E2 (27). Initially, an increased proliferative index with E2 treatment was established for the immortalized leiomyoma (UtLM-ht) cell line compared with the normal uterine smooth muscle (UtSM-ht) cell line (Fig. 1B). This increase in proliferation is blocked by treatment with ICI 182, 780 (Fig. 1B), thus indicating an ER-dependent process. The statistically significant level of increase in the rate of proliferation in leiomyoma cells treated with E2 is expected for cells of benign, smooth muscle origin. Although studies with animal models have found decreased levels of apoptosis in leiomyoma cells (62), findings from our laboratory are in agreement with others (20,21) that demonstrate higher rates of proliferation, rather than differences in apoptosis, are critical for leiomyoma growth (supplemental data Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online web site at http:// endo.endojournals.org).
In vivo xenograft studies whereby ovariectomized female nude mice were injected with either UtLM-ht or UtSM-ht cells and given blank or E2 pellets demonstrate that injected UtLM-ht cells form E2-responsive tumors that are larger than those formed by injection of UtSM-ht cells (Fig. 2A). Thus, we conclude that the proliferation rate observed in UtLM-ht cells with E2 is biologically significant.
E2 rapidly activates ERK1/2 in leiomyoma cells but not in normal uterine smooth muscle cells
Treatment of uterine leiomyoma cells with 1 nm E2 for 5 min results in a rapid increase in levels of phospho-ERK1/2 (Fig. 3, A and B). Similar results were observed with 0.1 and 10 nm E2 exposure (data not shown). ERK1/2 is the downstream effector of the MAPK cascade, which is typically activated in response to growth factor activation of receptor tyrosine kinases. Pretreatment with the ER antagonist ICI 182, 780 prevents this increase in phospho-ERK1/2 levels, thus demonstrating that E2’s rapid activation of ERK1/2 is an ER-dependent process (Fig. 3A). Treatment with agonists specific for ΕRα and ΕRβ, 1,3,5-Tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole, and 2,3-bis(4-hydroxyphenyl)-propionitrile, respectively, demonstrates that ERα is the primary contributing isoform to ERK1/2 activation in this cell type (supplemental data Fig. 2). Α rapid increase in ERK1/2 activation with 3 min E2 exposure is first detectable by Western blot analysis at 5 min but diminishes by 15 min (Fig. 3B). A second increase in phosphorylated ERK1/2 levels can be detected 16 h after rapid E2 treatment in leiomyoma cells (Fig. 3C). This second increase suggests that rapid E2 treatment can initiate longer-term MAPK activation that can contribute to proliferation.
However, unlike leiomyoma cells, normal uterine smooth muscle cells treated for 5 min with E2 did not demonstrate increased levels of phospho-ERK1/2 (Fig. 3A). Basal levels of ERK1/2 and PKCα are similar between the two cell lines (supplemental data Fig. 3, A and B).
E2 induces cAMP production and increased phospho-PKA levels in normal uterine smooth muscle cells but not leiomyoma cells, and this pathway inhibits ERK1/2 activation and proliferation
Treatment with 1 nm E2 results in a rapid increase in cAMP levels by 5 min in UtSM-ht cells but not UtLM-ht cells (Fig. 4, A and B). Moreover, levels of phospho-PKA also increase with 5 min E2 treatment in UtSM-ht cells (Fig. 4C). Increasing levels of cAMP results in activation of cAMP-dependent PKA, which in turn has been previously shown to interact with MAPK signaling (63,64,65). Inhibition of PKA with the pharmacological inhibitor H-89 results in increased levels of phospho-ERK1/2 in UtSM-ht treated with E2 (Fig. 4D). These data suggest that E2-induced PKA signaling has an inhibitory interaction with the MAPK cascade in UtSM-ht cells and may thus contribute to the low levels of phospho-ERK1/2 observed with E2 in this cell type. PKA has been shown by other laboratories to have an inhibitory effect on Raf activation (63,64,65). Interestingly, this pathway is not induced by E2 in the UtLM-ht cells that have high levels of phospho-ERK1/2 with E2 treatment.
Proliferation studies using the cAMP analog 8brcAMP, an active form of cAMP, demonstrate that cAMP signaling inhibits proliferation in both UtSM-ht and UtLM-ht cells (Fig. 5). Treatment of IBMX stabilizes cAMP from phosphodiesterase action and only UtSM-ht cells demonstrate reduced proliferative rates with IBMX and E2 treatment (Fig. 5). These results suggest that E2 initiates the production of cAMP, which in turn is stabilized by IMBX. This attenuated cAMP signal further inhibits proliferation of UtSM-ht cells with E2. Thus, the results of these experiments demonstrate that E2’s rapid activation of the cAMP-PKA pathway may be important in modulating MAPK activity and proliferation in normal uterine smooth muscle cells and that this pathway is not activated in leiomyoma cells.
E2 rapidly elevates phospho-PKCα levels in both normal uterine smooth muscle and leiomyoma cells and is dependent on the ER
Western blot analysis revealed that treatment of both UtLM-ht and UtSM-ht cells with E2 for 5 min results in significant increases in levels of phospho-PKCα (Fig. 6, A and B). Pretreatment of both UtSM-ht and UtLM-ht cells with the ER antagonist ICI 182, 780 (1 μm) prevented this increase in phospho-PKCα with E2, thus indicating an ER-dependent process (Fig. 6, A and B). Higher levels of phospho-PKCα are seen in UtLM-ht compared with UtSM-ht cells with E2 treatment (Fig. 6A). Phosphorylation of other PKC isoforms (δ, λ, θ, ζ) was not detected with a 5-min E2 incubation (data not shown). However, the possibility of PKCβ phosphorylation could not be eliminated due to the nature of the antibodies used.
PKCα promotes rapid ERK1/2 activation in leiomyoma cells but inhibits rapid ERK1/2 activation in normal uterine smooth muscle cells
Results from the following experiments demonstrate that PKCα differentially affects levels of ERK1/2 phosphorylation between leiomyoma and normal uterine smooth muscle cells. Our approach was to use a combination of both pharmacological and molecular tools to determine whether PKCα interacts with the MAPK cascade in this cellular system. Bis10 is a potent, selective inhibitor of PKC that inhibits PKC by interacting with its catalytic subunit (66), and Ro 31-8220 is a selective inhibitor of PKC at concentrations less than 1 μm (67). Pharmacological inhibition of PKCα in uterine leiomyoma cells results in a reduction of phospho-ERK1/2 levels with E2 treatment (Fig. 7A). Moreover, transfection of a constitutive-active PKCα resulted in increased levels of phosphorylated ERK1/2 compared with control transfected cells (Fig. 7B), thus confirming a positive relationship between PKCα and ERK1/2 activation that can then be disrupted by inhibitors of PKCα.
In contrast to leiomyoma cells, pharmacological inhibition of PKCα in normal uterine smooth muscle cells results in increased levels of phospho-ERK1/2 with E2 treatment (Fig. 8A). Transfection of normal uterine smooth muscle cells with a dominant-negative PKCα followed by exposure to E2 led to a rise in phospho-ERK1/2 levels (Fig. 8B). The reduction of active PKCα leading to an increase in phospho-ERK1/2 levels with E2 treatment implies that active PKCα inhibits phosphorylation of ERK1/2 in normal uterine smooth muscle cells.
Pretreatment of normal uterine smooth muscle cells with a pharmacological inhibitor of PKCα followed by rapid E2 treatment results in reduced cAMP levels compared with cells treated with E2 alone (Fig. 9). This reduction in cAMP signaling with PKCα inhibition suggests that these two pathways are not independent and PKCα’s contribution to cAMP-PKA signaling may be a potential mechanism of inhibition of MAPK signaling with E2’s rapid activation of PKCα in normal uterine smooth muscle cells.
Inhibition of conventional PKCs results in reduced proliferation in leiomyoma cells but increased proliferation in normal uterine smooth muscle cells
Pharmacological inhibition of PKC in leiomyoma cells led to reduced proliferation in response to E2 treatment (Fig. 10). In fact, PKC inhibition reduced E2-stimulated proliferation in leiomyoma cells to that observed in normal smooth muscle cells. This finding is in agreement with our observation that PKCα inhibition results in lowered levels of phosphorylated proliferation-associated ERK1/2 in leiomyoma cells.
On the other hand, pharmacological inhibition of conventional PKCs in normal uterine smooth muscle cells led to increased proliferation in response to E2 treatment (Fig. 10). Again, this data are in agreement with our finding that PKCα inhibition raises levels of phospho-ERK1/2, a proliferative signal, in E2-treated normal uterine smooth muscle cells.
Discussion
All experiments were conducted with immortalized UtSM-ht and UtLM-ht cells. Studies characterizing these cells have found that, unlike primary uterine leiomyoma cells (68), ER and progesterone receptor expression is maintained, thus allowing for the study of ER-dependent processes (69). In our laboratory, UtLM-ht cells express increased ER mRNA and protein levels compared with normal uterine smooth muscle cells (supplemental Fig. 4). This finding is consistent with other laboratories that have found increased levels of ERα in leiomyoma cells (70). Genomic signaling and E2 sensitivity in this leiomyoma cell line is intact and the use of these immortalized cell lines allows for the manipulation and study of rapid E2 signaling pathways.
In this study, we found differential rapid E2 signaling between the normal uterine smooth muscle and leiomyoma cells. Normal uterine smooth muscle is normally quiescent except during pregnancy. Our studies found that E2 does not promote ERK1/2 activation in our uterine smooth muscle cells but instead triggers pathways that are inhibitory to MAPK activation and proliferation. In contrast, we observe that these rapid E2 signaling pathways are altered or absent in leiomyoma cells to promote ERK1/2 activation and proliferation.
E2 triggers a rapid increase in levels of phosphorylated ERK1/2 via an ER-dependent mechanism in immortalized human uterine leiomyoma cells. ERK1/2 is the downstream effector of the proproliferative MAPK cascade that is typically activated by growth factors. Growth factor signaling pathways have been previously identified as being important to the expansion of leiomyomas. Leiomyomas may therefore be particularly susceptible to rapid effects of estrogens due to the integration of E2 and growth factor signaling (58,71,72,73). For example, previous findings from our laboratory (Martin M, Nierth-Simpson E, T.C. Chiang, and J.A. McLachlan JA, unpublished data) and others found that E2 treatment leads to the up-regulation of IGF-I (50,54,56,74). The findings of this study suggest that rapid actions of estrogens may increase the sensitivity of leiomyoma cells to proliferation by jump-starting growth factor loops, via the rapid activation of ERK1/2, that then sustain proliferation in leiomyoma cells.
The formation of cAMP in response to E2 in the uterus is one of the first described rapid E2 actions (75). We also found a rapid E2-induced increase in cAMP levels in normal uterine smooth muscle cells Increasing levels of cAMP triggers the activation of cAMP-dependent PKA, which has been shown to interact with the MAPK cascade (63,65). Depending on the cellular context, cAMP-PKA signaling may promote MAPK activation or inhibit Ras and Raf interaction (63,65). cAMP plays important roles in maintaining quiescence in uterine smooth muscle (76) and may also be important in reducing a proliferative response to estrogens because our study demonstrates that E2-induced cAMP-PKA signaling reduces ERK1/2 phosphorylation and proliferation in normal uterine smooth muscle cells. Although we demonstrate that cAMP signaling can reduce proliferation in leiomyoma cells, an increase in cAMP is not observed in leiomyoma cells with E2. The absence of this antiproliferative pathway may contribute to leiomyoma growth. Interestingly, cAMP signaling rapidly initiated by E2 may also suppress a transcriptional response to E2 because treatment of normal uterine smooth muscle cells with E2 and the PKA inhibitor H-89 results in significantly higher levels of E2-responsive progesterone receptor transcripts (supplemental Fig. 5). This finding is in agreement with a growing body of evidence that demonstrates that nongenomic/rapid signaling events can influence genomic events (77).
E2 has also been shown to differentially activate PKC isoforms (78,79) to influence proliferation in several cell lines (80). PKCα is a conventional PKC isoform that is similarly expressed between leiomyoma and normal uterine smooth muscle and, in addition to PKCζ, is the predominantly expressed isoform in uterine smooth muscle (81,82). Our data demonstrate that E2 rapidly increases levels of phospho-PKCα in both leiomyoma and normal uterine smooth muscle cells. Phosphorylation of the activation loop of PKCα is central to PKCα activity (83). When levels of Ca2+ and diacylglycerol rise, phosphorylated PKCα is recruited to the membrane for receptor-mediated targeting (84). Estrogens may affect PKCα activation via several mechanisms. First, we have shown that it can increase levels of phospho-PKCα. Second, we have found increased levels of active phospholipase C-β and -γ enzymes with E2 treatment (data not shown). Phospholipase C enzymes generate the Ca2+ and diacylglycerol necessary for full PKCα activation.
In this study, we report that E2-induced phospho-PKCα interacts with the proliferative MAPK cascade to inhibit phosphorylation of ERK1/2 in normal uterine smooth muscle cells. The effect of E2’s rapid increase in phospho-PKCα on inhibition of uterine smooth muscle proliferation may be 2-fold. First, we have shown that PKCα can inhibit ERK1/2 activation and that its promotion of cAMP-PKA signaling may be an important contributing mechanism to this inhibition. Second, it has been shown that PKC modifies ER binding by decreasing levels of cytosolic ER, thus lowering responsiveness to E2 in uteri (85). E2 has also been found to increase membrane-associated PKC isozyme expression in uterine muscle (86), which could further potentiate this antiproliferative effect. The inhibition of MAPK and cell growth with E2, although not generally observed in cells of epithelial origin, is not unique to uterine smooth muscle (87). Interestingly, it also occurs in another smooth muscle type: vascular smooth muscle (88).
Although we also observe an E2-induced increase in phospho-PKCα in leiomyoma cells, PKCα promotes ERK1/2 activation and proliferation in this cell type. PKCα has been shown to increase Ras activation (71,89,90) and can directly phosphorylate Raf (91), both of which are upstream of ERK1/2, in a variety of cell systems. Elevated levels of ERα may partially account for the greater levels of E2-induced phospho-PKCα, an ER-dependent process, in the leiomyoma vs. the normal uterine smooth muscle cells.
Interestingly, like cAMP-PKA signaling, PKCα also has important functions in uterine smooth muscle because it is involved in endothelin-1 and platelet-derived growth factor-induced ERK1/2 activation, human chorionic gonadotropin action (92), and growth in human and rat uterine smooth muscle cells during pregnancy (93,94). It has been proposed that uterine leiomyomas share characteristics with pregnant uterine smooth muscle cells (95), and a gene expression profile in an animal model shows that leiomyomas express several pregnancy-related genes (96). In this investigation, we demonstrate a mechanistic similarity between pregnant uterine smooth muscle and leiomyoma cells, PKCα’s interaction with the MAPK cascade.
This study presents a unique model system to investigate both mechanisms and biological impacts of rapid E2 signaling. Moreover, due to the noncancerous nature of leiomyomas, it allows for the study of pathways important to proliferation in the absence of neoplastic transformation. In conclusion, this report demonstrates that E2’s rapid activation of PKCα and cAMP signaling inhibits ERK1/2 activation in normal uterine smooth muscle cells and serves to prevent growth in this normally quiescent tissue (Fig. 11). However, this signaling becomes altered in the case of uterine leiomyoma disease such that an antiproliferative signal is either absent, as in the case of cAMP signaling, or is converted to a signal that promotes ERK1/2 activation and proliferation (PKCα signaling). Further investigation into upstream targets of E2 that result in the differential rapid signaling between normal uterine smooth muscle and leiomyoma cells should prove useful in finding a therapeutic target for the treatment of leiomyoma disease.
Supplementary Material
Acknowledgments
We thank Darlene Dixon, Ph.D., for the generous donation of the UtLM-ht and UtSM-ht cell lines. We also thank Barbara Beckman, Ph.D., for her donation of the constitutive-active and dominant-negative PKC plasmids. Lastly, we thank Syreeta Tilgman, Ph.D., Melyssa Bratton, Ph.D., and Ashley Fornerette for technical advice and manuscript revision.
Footnotes
This work was supported by Grant N000140611136 from the Office of Naval Research, Grant DK059389 from the National Institutes of Health, and a postdoctoral fellowship from the Louisiana Cancer Research Consortium.
Disclosure Summary: The authors of this manuscript have nothing to declare.
First Published Online January 29, 2009
Abbreviations: Bis10, Bisindolymaleimide × hydrochloride; 8brcAMP, 8-bromoadenosine-cAMP; E2, 17β-estradiol; ER, estrogen receptor; FBS, fetal bovine serum; IBMX, 3-isobutyl-1-methylxanthine; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PKA, protein kinase A; PKC, protein kinase C; UtLM-ht, Uterine leiomyoma; UtSM-ht, uterine smooth muscle.
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