A-Kinase Anchoring Protein 13 (AKAP13) Augments Progesterone Signaling in Uterine Fibroid Cells

Abstract

Context

Uterine leiomyomata (fibroids) are prevalent sex hormone‒dependent tumors with an altered response to mechanical stress. Ulipristal acetate, a selective progesterone receptor (PR) modulator, significantly reduces fibroid size in patients. However, PR signaling in fibroids and its relationship to mechanical signaling are incompletely understood.

Objective

Our prior studies revealed that A-kinase anchoring protein 13 (AKAP13) was overexpressed in fibroids and contributed to altered mechanotransduction in fibroids. Because AKAP13 augmented nuclear receptor signaling in other tissues, we sought to determine whether AKAP13 might influence PR signaling in fibroids.

Methods and Results

Fibroid samples from patients treated with ulipristal acetate or placebo were examined for AKAP13 expression by using immunohistochemistry. In immortalized uterine fibroid cell lines and COS-7 cells, we observed that AKAP13 increased ligand-dependent PR activation of luciferase reporters and endogenous progesterone-responsive genes for PR-B but not PR-A. Inhibition of ERK reduced activation of PR-dependent signaling by AKAP13, but inhibition of p38 MAPK had no effect. In addition, glutathione S-transferase‒binding assays revealed that AKAP13 was bound to PR-B through its carboxyl terminus.

Conclusion

These data suggest an intersection of mechanical signaling and PR signaling involving AKAP13 through ERK. Further elucidation of the integration of mechanical and hormonal signaling pathways in fibroids may provide insight into fibroid development and suggest new therapeutic strategies for treatment.

AKAP13 was bound to PRB and augmented progesterone-dependent gene activation via an ERK-dependent mechanism in uterine fibroid cells.

Uterine leiomyomata, or fibroids, are benign, sex steroid hormone‒dependent smooth muscle tumors that affect nearly 70% of white women and more than 80% of black women by age 50 years (1). Fibroids are estimated to add $5.9 to $34.4 billion annually to health care costs in the United States, including hospital admissions and surgeries, outpatient visits, medications, and lost work hours (2). Hysterectomy is the most common surgical intervention, but it is invasive and unsuitable for women who wish to preserve fertility. Other treatment alternatives such as myomectomy, MRI-guided focused ultrasound, cryomyolysis, and uterine artery embolization have been effective; however, each treatment modality has been associated with high recurrence rates as well as limitations and complications (3).

Published evidence supports a role of progesterone in the pathogenesis of uterine fibroids (4–15). Importantly, antiprogestins and selective progesterone receptor (PR) modulators such as ulipristal acetate (UPA) (16, 17), mifepristone (18–20), asoprisnil (21), and vilaprisan (22), used for treatment of heavy menstrual bleeding caused by uterine fibroids, also demonstrated a significant reduction in fibroid size. Specifically, UPA was shown to decrease fibroid volume, induce amenorrhea, and improve quality of life scores in randomized controlled trials (16, 23). Furthermore, a double-blind noninferiority trial showed that oral administration of 10 mg of UPA daily controlled bleeding in 98% of women and induced amenorrhea 2 weeks earlier than leuprolide acetate (24). Compared with placebo, UPA decreased fibroid volume by 21% in tumors ranging from 3 to 10 cm in diameter (17). Analysis of ex vivo fibroid samples suggested UPA acts to decrease production of extracellular matrix proteins, increase expression of metalloproteinases, and reduce cell proliferation (16, 25, 26), although the molecular details of selective PR modulator action in fibroids remain unclear (27).

One cardinal feature of fibroids is their altered extracellular matrix and increased stiffness (28–32). Mechanical stretch is thought to stimulate hypertrophy to prevent increased tension on the myometrium and maintain quiescence before delivery (33). We speculated that dysregulation of mechanical cues leading to myometrium hypertrophy under normal physiologic conditions may contribute to fibroid development (34). Fibroid development may in turn increase bulk, which further induces hypertrophy, potentially modulating gene expression through production of excessive extracellular matrix proteins, leading to altered mechanical signaling (35, 36). Transduction of mechanical signals often involves the Rho family of GTPases, a molecular switch regulating F-actin formation, maintenance of cell shape, and cell growth (37). Our group previously reported that a scaffold protein called A-kinase anchoring protein 13 (AKAP13; also called Brx or AKAP-Lbc), which contains a functional guanine nucleotide exchange factor domain (Rho-GEF) to activate RhoA, is highly upregulated in fibroids compared with matched myometrial tissues (38). Of note, an essential function of AKAP13 in mechanotransduction and cell survival has been described in human stem cells in culture (39). Paradoxically, uterine fibroids exist in an environment of increased mechanical stress but have a decreased response to mechanical cues (31, 38). Specifically, fibroid cells did not reorganize actin in response to mechanical strain. In addition to its action as a Rho-GEF, the carboxyl domain of AKAP13 was found to bind multiple steroid hormone receptors through a nuclear receptor interaction domain (NRID) located at its C-terminal (40, 41) and enhance ligand-dependent transcriptional activation by several nuclear hormone receptors, including estrogen receptor (ER) α (40), ERβ (41), and the glucocorticoid receptor (GR) (42). However, prior studies did not assess whether AKAP13 also interacts with the PR and modulates its transcriptional activity and downstream signaling.

Existing evidence suggests a possible interaction between mechanotransduction and steroid hormone signaling pathways in uterine fibroids, which may involve progesterone or the PR directly. The mechanism of this potential interaction is unknown, and it is not clear whether these pathways play a role in the mechanism by which UPA functions to decrease the size and symptoms of fibroids. We hypothesized that altered AKAP13 expression might represent a mechanism by which progestins and antiprogestins affect the pathophysiology of uterine fibroids. Further, we tested the hypothesis that mechanical signaling might be mediated by AKAP13, which affects hormone-dependent gene regulation in fibroid cells.

Materials and Methods

Tissue collection

Tissues were obtained from a randomized, placebo-controlled, double-blind clinical trial (NCT00290251) to evaluate the therapeutic effect of UPA on uterine fibroids with institutional review board approval. Premenopausal patients with symptomatic fibroids were recruited for treatment with UPA (10 or 20 mg/d) or placebo for a total of 3 months. Patients underwent hysterectomy at 90 to 102 days of treatment, and leiomyoma and myometrium tissue samples were collected (16).

Immunohistochemical staining

Paraffin-embedded patient-matched fibroid and myometrial tissues were sectioned (Histoserv, Inc., Germantown, MD) for immunohistochemical staining. Slides were deparaffinized in xylene (Sigma-Aldrich) and rehydrated with a series of sequential ethanol baths. Antigen retrieval was performed using sodium citrate buffer (pH, 6.0) heated to 95°C for a 45-minute incubation in a vegetable steamer. Sections were blocked for 30 minutes in 1X Tris-buffered saline containing 3% BSA/0.1% Tween-20 along with the appropriate serum. The addition of rabbit primary antisera was directed against AKAP13 at a 1:200 dilution. A peptide corresponding to the AKAP13 protein (CREKEKDKIKEKEKDS KEKEKDKKTLNGHTF) was used to generate polyclonal antiserum 6969 using standard techniques, and binding to BRX was confirmed using an enzyme-linked immunosorbent assay (Covance Laboratories, Sterling, VA) (43). For human PR-A and PR-B, antibody was obtained from Cell Signaling Technology (clone: 6A1), used at a 1:200 dilution. For anti-human PB-B‒specific antibody, antibody was obtained from Cell Signaling Technology (clone: C1A2), used at a 1:200 dilution. Normal breast tissue served as a positive control for AKAP13. Negative controls included secondary antibody only, without the addition of the primary antibody or preimmune sera. After washing, slides were incubated in biotinylated anti-rabbit/mouse IgG (Vector Laboratories, Burlingame, CA) and then Elite™ ABC reagent (avidin-biotin peroxidase complex). The peroxidase was then developed with the addition of 3,3′-diaminobenzidine peroxidase substrate. Sections were counterstained with hematoxylin, dehydrated in ethanol, and mounted in Permount for microscopic examination. Pictures were taken at 20× magnification. The scale bar represents 1 μm.

Plasmids

Progesterone-response element-luciferase reporter (PRE-luc) and the basal promoter system TA-luciferase were purchased from Signosis (Santa Clara, CA). MMTV-luciferase reporter (MMTV-luc; luciferase gene under the control of the progesterone-responsive murine mammary tumor virus promoter), was generously provided by Dr. Tomoshige Kino (Sidra Medical and Research Center, Qatar). Expression plasmids for the 1.8-kb human AKAP13 were previously described (41). Human PRs (PR-A and PR-B) expressing plasmids were generously provided by Dr. Stoney Simons [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health (NIH), Bethesda, MD]. Empty expression vectors pcDNA4-HisMax and pSG5 (Invitrogen, Carlsbad, CA, and Agilent Technologies, Santa Clara, CA, respectively) were used as negative controls for AKAP13 and PR, respectively.

Cell culture

An immortalized patient-derived uterine fibroid cell line (P51F) was developed by Malik et al. (44, 45). Dr. Malik kindly provided the cell line to us. P51F cells were maintained in DMEM-F12 (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; HyClone) and 1X Normocin (InvivoGen) at 37°C and 5% CO₂. Fibroid cells are known to express endogenous PR and AKAP13 (38, 46). We used P51F cells for PRE-luc studies. African green monkey kidney COS-7 cells were obtained from American Type Culture Collection (Manassas, VA) and grown in DMEM supplemented with 10% FBS, 100 U of penicillin, 1 μg/mL of streptomycin sulfate, and 2 mM of glutamine (Thermo Fisher, Waltham, MA). We used COS-7 cells for PRE-luc and MMTV-luc studies because they do not express endogenous PR and AKAP13. Human breast carcinoma T47D cells were also obtained from American Type Culture Collection and grown in RPMI1640 with the same supplements mentioned previously. We used T47D for expression of alkaline phosphate, a PR-dependent gene, for progesterone stimulation (47–49). T47D cells express AKAP13 endogenously.

Transient transfection

After plating, when cells achieved ∼50% confluency, culture media were replaced with phenol red free, 10% charcoal-stripped FBS. After 24 hours, the cells were ready for transfection. Transfection was performed with FuGENE 6 (Roche Applied Science, Indianapolis, IN) or HilyMax5 (Dojindo, Rockville, MD) per the manufacturer’s directions. Each well of cells was cotransfected with 0.5 μg of reporter plasmid (PRE-luc or MMTV-luc), 0.5 μg of AKAP13, and 25 ng of PR-A or PR-B expression plasmids. Total DNA was kept constant by the addition of empty vector (pcDNA4-HisMax or pSG-5). Twenty-four hours after transfection, 40 nM of progesterone or ethanol vehicle was added to the media. After 24 hours of progesterone treatment, cells were lysed with a reporter lysis buffer (Promega, Madison, WI), and luciferase activities were determined by using the Firefly luciferase kit (Promega) and Moonlight luminometer (Promega). Luciferase activities were normalized by protein quantification assay (Bradford; BioRad, Hercules, CA).

AKAP13 knockdown by siRNA transfections and alkaline phosphatase assay

To test whether AKAP13 would influence an endogenous PR-responsive gene, we examined effects on the de novo synthesis of alkaline phosphatase (AP), a known progesterone-responsive gene in T47D cells. T47D cells are a commonly used cell line because of their robust, dose-dependent and time-dependent response to progesterone stimulation (48, 49). T47D cells (4 × 10⁴ per well) were plated in 48-well plates in DMEM/F12 containing 2% charcoal-stripped FBS. Upon attaining 30% to 40% confluence, cells were transfected with AllStars Negative Control (Qiagen) or AKAP13 small interfering RNA (siRNA) (5′ GAGAAUGCAGAACGUUUPAATT 3′; Millipore Sigma, Burlington, MA) using Lipofectamine 2000 per the manufacturer’s recommendations. Two days after transfection with siRNAs, cells were treated with increasing concentrations of progesterone or vehicle. After incubation for 12 hours, cells were washed with 1X Tris-buffered saline, and 100 μL of 0.1M Tris-HCl (pH, 9.8) containing 0.2% Triton X-100 was added to each well. Cells were lysed by shaking for 15 minutes. The substrate [300 μL of 0.1M Tris-HCl (pH, 9.8) containing 4 mM of p-nitrophenyl phosphate] was added per well. Absorbance readings at 405 nm were measured on a Bio-Rad Benchmark Plus microplate spectrophotometer. Representative data with SEM of triplicate are shown. The experiment was repeated twice with similar results.

Glutathione S-transferase‒binding studies

A 1.8-kb fragment of AKAP13 and an EcoRI fragment corresponding to the C-terminal region of AKAP13 was subcloned in-frame into the bacterial expression plasmid pGEX2TKB as previously described (40). Two AKAP13 chimeras were created: the longer glutathione S-transferase (GST)-AKAP13 construct (FT) and the C-terminal fragment (R5) containing the NRID. Recombinant GST-0 control proteins (empty nonchimeric glutathione transferase) GST-AKAP13 (FT and R5) were generated from bacterial plasmids and immobilized on sepharose beads per manufacturer’s instructions (Pfizer, New York, NY).

To radiolabel the PR, a fragment of PR corresponding to amino acids 687 through the C-terminus was created via PCR amplification of the full-length human PR-B open reading frame from pSG5-hPRB bacterial plasmid, and in vitro transcription and translation (TNT kit; Promega) were performed in the presence of ³⁵S-labeled methionine. This fragment, designated PR687C, is common to all PR isoforms and contains the AF-2 activation function of the protein as well as the ligand-binding domain.

Next, 100,000 cpm of ³⁵S-labeled PR protein per binding reaction was incubated with GST beads at 4°C in Hepes binding buffer media containing 40 mM HEPES, 75 mM KCl, 0.5 mM EDTA, 5 mM MgCl₂, 1 mM dithiothreitol DTT, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, bovine serum albumin 0.5 mg/mL, and 0.05% nonidet P40. After five wash steps in Hepes binding buffer, bound proteins were eluted in sample buffer and analyzed by SDS-PAGE and autoradiography. Then 10,000 cpm was loaded for each input lane. In vitro synthesized luciferase protein, which does not bind to AKAP13, served as a negative control.

MAPK inhibition studies

African green monkey kidney (COS-7) cells were cultured and transfected as described previously. Cells were pretreated with p38 MAPK inhibitors [2 μM of SB202190 or 10 μM of SB203580 in dimethyl sulfoxide (DMSO)] (50) or extracellular ERK inhibitor (51) (0.1, 1, or 10 μM of U0126 in DMSO) before addition of the hormone ligand. All inhibitors were purchased from Millipore (Millipore-Sigma, Burlington, MA). DMSO was used for vehicle controls. After pretreatment, progesterone was added to a final concentration of 40 nM for 24 hours. Finally, cells were lysed and assayed as described previously. To validate the activity of each p38 MAPK inhibitor COS-7 cells were treated with inhibitor or DMSO as described previously, then shocked with 50% sorbitol to induce phosphorylation of p38 MAPK (52). Cell lysates were extracted and resolved via SDS-PAGE. Western blotting was performed on polyvinylidene difluoride (PVDF) membranes (Millipore-Sigma, Washington, DC) probed with anti-p38 MAPK antibodies [phospho-p38 MAPK (Thr180/Tyr182) (D3F9), total p38 MAPK (D13E1); both from Cell Signaling Technology, Danvers, MA]. Immune complexes were detected using enhanced chemiluminescence (Thermo Fisher Scientific) per manufacturer’s directions.

Statistical analysis

All experiments were repeated three to seven times as described in the figure legends. Data between groups were compared using an unpaired two-tailed Student t test using Prism 5 software (GraphPad Software, Inc., La Jolla, CA). Statistical significance was reported if P < 0.05.

Results

UPA altered AKAP13 and PR expression in fibroid tissues

To explore whether UPA altered the expression level of AKAP13 in fibroid tissue, we examined tissues harvested from UPA-treated (or placebo) subjects. Immunohistochemical stains showed that samples from patients treated with UPA contained lower AKAP13 levels than samples of placebo tissues (Fig. 1A). This observation raised the possibility that one action of UPA relieving fibroid burden might be due, at least in part, to modulation of AKAP13. We also evaluated the expression of PR (targeting both PR-A and PR-B) in the same placebo and UPA-treated samples. We found that PR expression was also reduced in UPA-treated samples compared with placebo samples, as expected (Fig. 1B). We conducted additional staining using a PR-B‒specific antibody. We observed reduction of PR-B expression in samples from patients treated with UPA (Fig. 1C). The reduction in AKAP13 and its known role in mechanical signaling, coupled with the reduction in fibroid size caused by UPA, led us to probe the possibility of a direct relationship between progesterone signaling and AKAP13.

Figure 1.

Immunohistochemical (IHC) staining showed altered expression of AKAP13 and PR in ulipristal acetate‒treated fibroids and myometrium. Fibroid tissues were collected from patients who received placebo or ulipristal acetate treatment (20 mg per dose). Tissues were stained with (A) antihuman AKAP13 antibody, (B) antihuman PR antibody (PR-A and PR-B), or (C) antihuman PR-B‒specific antibody to show the abundance of these proteins in these tissues. (A) Black arrowheads show the intense staining of AKAP13 in placebo samples but its disappearance in ulipristal acetate‒treated samples. Images of two representative patients from each group are shown. Six patients from each group were evaluated with similar results. Magnification = 20×. Scale bar represents 1 μm.

AKAP13 enhanced ligand-dependent PR transcriptional activities

Given the change in AKAP13 with antiprogestin treatment and evidence that AKAP13 enhanced ligand-dependent transcriptional activation by other nuclear hormone receptors, including ERα, ERβ, and GR (41, 42, 53, 54), we assessed whether AKAP13 modulated PR action in fibroid cells. To test this possibility, an immortalized uterine leiomyoma cell line developed from human surgical specimens, named P51F, was transfected with a PR-B expression vector with or without a cotransfected AKAP13 expression vector. We used progesterone response element PRE-luc, which contains three repeats of consensus progesterone response element (PRE) (55). When fibroid (P51F) cells were transfected with only PR-B, there was a significant fivefold progesterone-dependent induction of PRE-luc activity, as expected (Fig. 2A). Of note, coexpression of AKAP13 further enhanced the ligand-dependent activation to 10-fold (Fig. 2A). Next, we repeated the experiments in COS-7 cells, which do not express endogenous PR or AKAP13. We observed that AKAP13 enhanced the progesterone-induced PRE-luc activity by 12-fold compared with basal activity (Fig. 2B).

Figure 2.

Progesterone (P4)-dependent gene activation by PR-B is augmented by expression of AKAP13. (A) Human fibroid cells derived from immortalized human fibroid tissues (P51F) and (B) cells from the monkey kidney cell line COS-7 were cotransfected with plasmids of PRE-luc and human PR-B. The cotransfection of AKAP13 and the addition of P4 (40 nM) show augmented PRE-luc activity. (C) COS-7 cells were cotransfected with PRE-luc and either PR-A or PR-B. (D) COS-7 cells were cotransfected with MMTV-luc, PR-B, and AKAP13 as indicated by plus (+) or absence (−). Luciferase activity was determined by luminometer and normalized by protein concentration in each well. The fold-changes were calculated by normalization with luciferase value in wells without AKAP13 transfection and no P4 treatment. Each condition was repeated in triplicate. Each graph represents the experimental data with SEM of five to seven experiments with similar results. Statistical analysis was done using the Student t test. *P < 0.05; **P < 0.01; ***P < 0.001. ns, no statistical significance.

To determine the regions of PR required for the effect, we also tested whether AKAP13 augmented PR-A‒mediated PRE-luc response. PR-A and PR-B are encoded from the same gene, but they bind different sets of coregulators and have different posttranslational modification, which results in their different capabilities to regulate genes diversely (56). We transfected COS-7 cells with the same amount of PR-A or PR-B expression plasmids. We observed that PR-B, but not PR-A, induced progesterone-dependent activation of the PRE-luc reporter (Fig. 2C). The induction was similar in both P51F and COS-7 cells (data not shown). This result indicates that PR-B was solely responsive to AKAP13 in this system. A previous study also showed that PR-B, but not PR-A, was responsible for breast cancer cell migration (57). The posttranslational modification of PR in these cells may contribute to the effect.

Next, we tested another luciferase reporter driven by the native promoter MMTV (MMTV-luc) that contains PRE (58). Results showed that expression of AKAP13 significantly enhanced the transcription of progesterone-mediated MMTV promoter from threefold to 15-fold (Fig. 2D). We also conducted control experiments using the basal promoter of PRE-luc (which contains only TATA box sequence, so-called TA-luc) and MMTV-luc (contains the minimal promoter of thymidine kinase, so-called TK-luc). As expected, AKAP13 did not increase the activity of these control luciferase reporters (data not shown). Taken together, these findings suggest that overexpression of AKAP13 augments the ligand-dependent action of PR-B but not PR-A in cotransfection assays in the two cell lines.

AKAP13 knockdown decreased the activation of an endogenous progesterone-responsive gene

Next, we asked whether AKAP13 would influence an endogenous PR-responsive gene. To test this, we examined effects on the de novo synthesis of AP, a known progesterone-responsive gene in T47D cells. T47D cells were used for this experiment because of their well-described dose-dependent response to progesterone stimulation (48, 49). T47D cells express AKAP13 endogenously (47) (unpublished); therefore, they serve as a good model for a knockdown study. Using siRNA directed against AKAP13, we reduced the expression of AKAP13 by at least 50% compared with scrambled siRNA. At 1 nM of progesterone treatment, there was a significant increase in AP activity in cells transfected with control siRNA (Fig. 3). The AP activity in control cells was maximal by 10 to 40 nM of progesterone. We observed a 50% reduction in AP activity from 5 to 20 nM of progesterone in cells transfected with AKAP13 siRNA (Fig. 3). There are limitations because these experiments used T47D cells. Effects observed in breast cancer cells may not be transferable to a uterine fibroid cell line. Nonetheless, these data suggest that AKAP13 may be necessary for maximal activation by PR of an endogenous progesterone-responsive gene.

Figure 3.

AKAP13 knockdown by siRNA reduced expression of a progesterone (P4)-responsive gene. The human breast cancer cell line T47D was transfected with siRNA targeting AKAP13 or nontargeting siRNA negative control. Two d after transfection, cells were treated with the indicated dose of P4. Twelve h after P4 treatment, the expression of the P4-responsive gene alkaline phosphatase was measured by absorbance at 405 nm. Representative data with SEM of triplicate are shown. The experiment was repeated twice with similar results. Statistical analysis was done using the Student t test. **P < 0.01; ***P < 0.001.

AKAP13 activated PR partially through an ERK-dependent pathway

The AKAP family serves as a signaling hub to integrate and regulate spatial and temporal crosstalk between multiple signaling pathways (59). We previously showed that AKAP13 mediated crosstalk of GR via a p38 MAPK (54). Likewise, AKAP13 augmented the ligand-dependent activity of ERβ via a p38 MAPK-dependent mechanism (41). Therefore, we investigated whether the progesterone ligand‒induced PR-B transactivation involved crosstalk with the p38 MAPK pathway. Under our standard transfection conditions in COS-7 cells using MMTV-luc, p38 MAPK inhibitors (SB202190 and SB203580) successfully inhibited phosphorylation of p38 MAPK (Fig. 4A). However, the inhibitor caused no significant changes in progesterone-induced activation of MMTV-luc (Fig. 4B). These data suggest that p38 COS activity is not required for augmentation of PR activity by AKAP13.

Figure 4.

Augmentation of PR-B activity by AKAP13 was affected by the inhibition of the ERK pathway and not by the p38 MAPK pathway. (A) COS-7 cells were treated with p38 inhibitor SB203580 (10 μM) or SB202190 (2 μM) for 1 h. Cell lysate was harvested, and the quantification of phospho-p38 MAPK (active) and total p38 MAPK was done by western analysis using specific antibodies. (B) COS-7 cells were cotransfected with MMTV-luc reporter plasmid, human PR-B, and human AKAP13. Twenty-four h after transfection, the p38 inhibitors SB203850 and SB202190 were added to the cell culture for 1 h before progesterone (P4) treatment. (C) COS-7 cells were treated with the ERK inhibitor U0126 (0.1, 1, and 10 μM) for 1 h before P4 treatment. Cell lysate was collected 24 h after P4 treatment. Luciferase activity was determined by luminometer and normalized by protein concentration in each well. MMTV-luc activity in cells transfected with AKAP13 without any inhibitors was designated to be 100%, and the ERK inhibitor, U0126, was added in increasing concentrations as noted by the black triangles. Each condition was repeated in triplicate. Presence (+) or absence (−) of factor, as noted. Each graph represents data with the SEM of five to seven experiments with similar results. Statistical analysis was done using the Student t test. **P < 0.01.

Next, we tested whether enhancement of progesterone-dependent activity of PR-B by AKAP13 required activation of the ERK pathway. Pretreatment with the ERK inhibitor U0126 resulted in reduction in AKAP13 activation of PR by 40% at 1 μM, which increased to 20% at 10 μM (Fig. 4C). This suggests that ERK activity is partially required for enhancement of progesterone-dependent activity of PR-B by AKAP13.

AKAP13 bound to the C-terminal of PR

Our prior work revealed direct interactions of AKAP13 with the ligand-binding regions of ERα, ERβ, and GR through the C-terminal region of AKAP13. Therefore, we tested whether a C-terminal fragment of human PR-B (residues 687 to 933, named PR687C) bound to AKAP13 in GST‒pull-down experiments (Fig. 5A). Results of the pull-down assay demonstrated that PR687C bound to both the longer form of AKAP13 (GST-FT; Fig. 5B, lane 3) as well as the smaller NRID-containing carboxyl fragment (GST-R5; Fig. 5B, lane 4). PR687C did not bind to GST-0 (control protein; Fig. 5C, lane 2). In addition, control experiments with radiolabeled luciferase protein instead of PR-B did not reveal binding of luciferase to either GST-0 or GST-FT (Fig. 5C). Positive control experiments using radiolabeled ER confirmed binding of AKAP13 with ER as previously reported by our group (60) (data not shown). These results suggest a direct and specific interaction between AKAP13 and PR in vitro, most likely involving the NRID of AKAP13.

Figure 5.

Interaction between AKAP13 and PR. (A) Upper panel shows the schematic diagram of two human AKAP13-GST fusion proteins used for the pull-down assay: the 1.8-kb version (GST-FL) and the C-terminal version (GST-R5). Both versions contain the NRID domain that was previously shown to bind ER. GST-0 is the GST-his tag alone without any fusion. Lower panel shows that the C-terminal version of human PR protein (PR687C) was ³⁵S-labeled and used in this study, as shown. (B) Labeled PR-B C-terminus (PR687C) protein was incubated with various fusion proteins. Lane 1, ³⁵S-labeled PR-B input. Lane 2, GST-0 did not precipitate labeled PR-B. Lane 3, GST-FL AKAP13 did precipitate labeled PR-B (black arrow). Lane 4, the NRID region of AKAP13 in GST-R5 precipitated labeled PR-B. (C) Luciferase was ³⁵S-labeled (Lane 1, input) and incubated with GST-FL (Lane 2) or GST-0 (Lane 3) to serve as a negative control for AKAP13 binding. No binding was observed (open arrow).

Discussion

This study explored the role of AKAP13, a scaffold protein with Rho-GEF activity with a known role in mechanotransduction, and its influence on ligand-dependent PR activity in fibroids. We found that coexpression of AKAP13 greatly augmented ligand-dependent PR-B transactivation activity. Experiments suggested that ERK was partially involved in the AKAP13-mediated upregulation of PRE activity. Finally, we showed a direct interaction of PR with AKAP13 through the C-terminal region, which contains the NRID, a conserved motif that has been shown to bind other hormone receptors. In Fig. 6, we propose a working model based on our in vitro data that highlights the integration of AKAP13 with progesterone action. As a scaffold protein, AKAP13 is capable of bringing in close proximity the molecules involved in steroid hormone signaling (PR), mechanotransduction (Hippo/YAP), and the ERK pathways, which ultimately may contribute to the pathogenesis of fibroids through increased extracellular matrix (ECM) production, decreased ECM degradation, and increased growth.

Figure 6.

Proposed model of AKAP13 and PR-B signaling in fibroids. Our working model suggests that AKAP13 augments ligand-dependent activity of PR-B through ERK, possibly through close interaction of PR-B with AKAP13. Because AKAP13 is an essential regulator of the transcription factor YAP in mechanotransduction, it may potentially regulate PR activity through crosstalk with the Hippo pathway. Red = actin filament; blue box = integrin complex. VEGF, vascular endothelial growth factor. AKAP13 activates Rho (+) and is capable of augmenting ligand-dependent PR activity (+). PR then may affect gene transcription. The involvement of AKAP13 in both processes suggests integration of mechanical and progesterone signaling. Arrows suggest possible relationships of the factors in the pathway.

Our study showed that AKAP13 expression in fibroid tissue was affected by UPA treatment and that AKAP13 augmented PR-B action. UPA, a specific antagonist of PR, has been implicated in different pathways as achieving reduction of fibroid size. First, UPA affects the expression of soluble growth factors and angiogenic factors in vitro. Ciarmela et al. (61) and Islam et al. (62) reported that UPA decreases the expression of activin. Xu et al. (63) showed that UPA downregulated expression of vascular endothelial growth factor and adrenomedullin. In addition, UPA induces apoptosis of fibroid cells by increasing caspase-3 activity and decreasing antiapoptotic Bcl-2 expression (64).

AKAP13 is known to play a role in mediating steroid hormone signaling. We previously showed that AKAP13 interacted with GR and mediated tissue-specific responses to stress (42). Specifically, Kino et al. (42) showed that lymphocytes isolated from AKAP13 haplo-insufficient mice exhibited a significantly lower sensitivity to glucocorticoids. Further work showed that the signaling is important for osmotic homeostasis (54). AKAP13 serves as a convergence point of p38 MAPK, NFAT, and JIP4 to mediate optimal GR signaling (54). Similarly, the current study revealed the integration of ERK and PR-B signaling through AKAP13, which could shed light on the unsolved problem of functional withdrawal of PR in humans during parturition (65). Overexpression of AKAP13 greatly increased the transcriptional activity of ER (over sixfold induction) (40). Collectively, these data raise the possibility that AKAP13 plays a role in modulating steroid hormone signaling that contributes to fibroid pathology.

Fibroids are known for their increased stiffness compared with neighboring myometrial tissues (31, 34). Our group and others have provided evidence that fibroids exhibit an altered mechanical homeostasis (29, 31, 34, 38, 66). Altered mechanical signaling may be influenced by the altered extracellular matrix of uterine fibroids (35, 36). Downstream cellular processes translate mechanical stimuli into biochemical signals, allowing cells to adapt to their physical environment by way of force transmission between the extracellular matrix, the cytoskeleton, and the nucleus (67). Cells sense force through mechanosensitive proteins, resulting in the opening of membrane channels, alterations in binding affinities, and increased phosphorylation, which serve to activate downstream signaling pathways (67). Altered mechanical signaling is responsible for the pathogenesis of medical conditions, including fibroids, cardiac hypertrophy, and some neoplasms (68). In addition to the potential role of AKAP13 as a convergent point of hormonal signaling, as discussed earlier, these new data suggest that AKAP13 also may play a central role in integrating crosstalk between mechanical clues and hormonal signaling.

Through understanding of the molecular interaction of PR-B and AKAP13, this study may suggest additional therapeutic options for fibroids. Combination therapy is a viable and logical option to improve the therapeutic effects of UPA. For example, a recent presentation by Malik et al. (69) showed that treatment with UPA and simvastatin (to treat hyperlipidemia) synergistically reduced collagen expression, inhibited proliferation, and increased apoptosis in a patient-derived leiomyoma cell line and fibroid stem cells. Moreover, tranilast, an antiallergic compound, and liarozole, a retinoic acid‒metabolizing blocking agent, are thought to reduce fibroid ECM by reducing profibrotic factors such as TGF-β and activin-A (70, 71). Profibrotic factors that exacerbate the accumulation of and unbalanced production of matrix-modeling proteins are key to this pathogenesis mechanism for fibroids (72). In addition, lysophosphatidic acid is known to activate the activity of AKAP13 through Gα₁₂ and Gα₁₃ (73). It has been shown that LPA influences growth of fibroids in vitro with increased cell proliferation (74). These are also potential future targets for fibroids in conjunction with UPA treatment.

In this report, we examined fibroid cells and tissues that were developed; however, it is possible that disordered progesterone signaling may have a greater role earlier in fibroid development (14, 15). Whitaker et al. (26) observed opposite effects on PR expression in the endometrium with UPA treatment. They observed increased PR compared with the secretory phase control and no change in the proliferative phase (26). We suspect that this difference is specific to the endometrium or possibly the dose of UPA or duration of treatment.

In conclusion, our results provide a rationale for pursuing further characterization of AKAP13 as a molecule of interest in uterine fibroid pathophysiology. Our data suggest that fibroid formation is associated with increased expression of AKAP13, which could promote a progesterone-mediated fibroid response and, conversely, may explain a mechanism by which UPA works to decrease uterine fibroid growth. We recognize that further in vivo studies are needed to confirm these findings in the in vitro system. Additional characterization of the altered states of mechanotransduction in uterine fibroids may elucidate the molecular mechanism of this common disease.

Glossary

Abbreviations:

AKAP13

A-kinase anchoring protein 13

AP

alkaline phosphatase, DMSO, dimethyl sulfoxide

ECM

extracellular matrix

ER

estrogen receptor

FBS

fetal bovine serum

GR

glucocorticoid receptor

GST

glutathione S-transferase

MMTV

murine mammary tumor virus

MMTV-luc

MMTV-luciferase reporter

NRID

nuclear receptor interaction domain

PR

progesterone receptor

PRE

progesterone response element

PRE-luc

progesterone-response element-luciferase reporter

Rho-GEF

guanine nucleotide exchange factor

siRNA

small interfering RNA

TIMP

tissue inhibitor of metalloproteinase

UPA

ulipristal acetate